Received: 25 August 2022

| Accepted: 12 July 2023

DOI: 10.1111/jpy.13373

RESEARCH ARTICLE

Gene-­rich plastid genomes of two parasitic red algal

species, Laurencia australis and L. verruciformis
(Rhodomelaceae, Ceramiales), and a taxonomic revision
of Janczewskia

Maren Preuss1,2 | Pilar Díaz-­Tapia3,4 | Heroen Verbruggen5 |

Giuseppe C. Zuccarello2

1
National Institute of Water and
Atmosphere Research, Wellington,
New Zealand
2
School of Biological Sciences, Victoria
University of Wellington, Wellington, New
Zealand
3
Coastal Biology Research Group, Faculty
of Sciences and Centre for Advanced
Scientific Research, University of A
Coruña, A Coruña, Spain
4
Instituto Español de Oceanografía
(IEO-­CSIC), Centro Oceanográfico de A
Coruña, A Coruña, Spain
5
School of BioSciences, University of
Melbourne, Parkville, Victoria, Australia
Correspondence
Maren Preuss, National Institute of
Water and Atmosphere Research,
Wellington 6021, New Zealand; School of
Biological Sciences, Victoria University of
Wellington, PO Box 600, Wellington 6140,
New Zealand.
Email: maren.preuss@niwa.co.nz
Funding information
Australian Biological Resources
Study, Grant/Award Number: Activity
4-­G046WSD; Royal Society Te Apārangi,
Grant/Award Number: Marsden Fast-­
Start/ 19-­VUW-­0 06; Xunta de Galicia,
Grant/Award Number: ED481D/ 2017/011/
03IN858A2019-­1630129
Editor: M. Oliveira
Abstract
Parasitic red algae are an interesting system for investigating the genetic
changes that occur in parasites. These parasites have evolved independently
multiple times within the red algae. The functional loss of plastid genomes
can be investigated in these multiple independent examples, and fine-­scale
patterns may be discerned. The only plastid genomes from red algal parasites
known so far are highly reduced and missing almost all photosynthetic genes.
Our study assembled and annotated plastid genomes from the parasites
Janczewskia tasmanica and its two Laurencia host species (Laurencia elata
and one unidentified Laurencia sp. A25) from Australia and Janczewskia ver-
ruciformis, its host species (Laurencia catarinensis), and the closest known
free-­living relative (Laurencia obtusa) from the Canary Islands (Spain). For
the first time we show parasitic red algal plastid genomes that are similar in
size and gene content to free-­living host species without any gene loss or
genome reduction. The only exception was two pseudogenes (moeB and
ycf46) found in the plastid genome of both isolates of J. tasmanica, indicating
potential for future loss of these genes. Further comparative analyses with the
three highly reduced plastid genomes showed possible gene loss patterns, in
which photosynthetic gene categories were lost followed by other gene cat-
egories. Phylogenetic analyses did not confirm monophyly of Janczewskia,
and the genus was subsumed into Laurencia. Further investigations will de-
termine if any convergent small-­scale patterns of gene loss exist in parasitic
red algae and how these are applicable to other parasitic systems.
KEYWORDS
Janczewskia, Laurencia, moeB, obligate hemiparasites, parasite origin, phylogenomics,
pseudogenes, red algal parasites, reductive plastid evolution, ycf46

I N TROD UC T I O N in nearly all organisms with photosynthetic ancestors,

Comparative plastid genome studies in parasitic sys-
tems are important for understanding evolutionary
changes during the transition from a free-­living to a
parasitic life strategy. Plastid genomes are observed
and besides photosynthetic capability, these organ-
elles provide many other functions including amino
acid, fatty acid, and lipid synthesis (Wise, 2007). In
parasites, plastid genomes commonly lose genes,
and in many cases, highly reduced plastids have

Abbreviations: CTAB, cetyltrimethylammonium bromide; HTS, high-­throughput sequencing.

950 | © 2023 Phycological Society of America. wileyonlinelibrary.com/journal/jpy Journal of Phycology. 2023;59:950–962.


PLASTID EVOLUTION IN JANCZEWSKIA
lost their photosynthetic capability (e.g., de Koning &
Keeling, 2006; Kayama et al., 2020). Functional and
physical reductions in plastid genomes occur over
time, and the degree of degradation in parasitic plastid
genomes reflects the transition from autotrophy to par-
asitism, where organisms with intact plastid genomes
are less reliant on their hosts (for nutrition) than par-
asites with highly reduced plastid genomes (Banerjee
et al., 2022; Wicke & Naumann, 2018). The early stages
of this transition are of interest for identifying any pat-
terns of gene loss that may be common among para-
sites. Fine-­scale patterns in recently diverged parasites
are hard to investigate, as few study systems provide:
(i) multiple independently evolved parasites in order to
observe different stages of gene and functional loss
and (ii) comparisons with closely related free-­ living
organisms.
Parasitic red algae are excellent systems for investi-
gating these transitions, as these parasites evolved in-
dependently multiple times. These parasites are highly
diverse with almost 130 species currently described
(Freese & Lane, 2021; Preuss et al., 2017; Preuss &
Zuccarello, 2018; Preuss & Zuccarello, 2019). Molecular
phylogenetics show different evolutionary relationships
of these parasites to their hosts (e.g., Goff et al., 1996;
Kurihara et al., 2010; Preuss & Zuccarello, 2018). Most
parasites are closely related to their hosts, often group-
ing with the host as its closest relative, indicating that
the parasite evolved recently (Goff et al., 1997; Ng
et al., 2014). In other cases, the parasites are phylo-
genetically distant from their hosts, and their closest
relative is a non-­host species (Goff et al., 1996; Kuri-
hara et al., 2010; Preuss & Zuccarello, 2018; Zuccarello
et al., 2004).
The discovery of the highly reduced plastid genome
in the unpigmented parasite Choreocolax polysipho-
niae (Salomaki et al., 2015) demonstrated that parasitic
red algae can retain their own plastid genome instead
of only maintaining the acquired plastid genome of their
host (Goff & Coleman, 1995). Shortly after that study,
two more highly reduced plastid genomes were identi-
fied in Harveyella mirabilis (Salomaki & Lane, 2019) and
Pterocladiophila hemisphaerica (Preuss et al., 2020).
Comparative plastid genomic analysis showed the loss
of almost all photosynthetic genes (with the exception
of petF) and a conserved core of housekeeping genes.
To date no further full plastid genomes of parasitic red
algae have been sequenced. Further studies will be im-
portant for revealing the extent of the changes seen in
parasite plastid genomes and if there are any shared
patterns that can be linked to the change from free-­
living to parasite.
The parasitic genus Janczewskia was described
based on J. verruciformis growing on Laurencia ob-
tusa in Naples, Italy (Guiry & Guiry, 2022; Solms-­
Laubach, 1877). Currently, a total of 11 Janczewskia
species are taxonomically accepted, and these mostly
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    | 951
grow on Laurencia spp. and occasionally on closely re-
lated genera, such as Chondria and Osmundea (Pre-
uss et al., 2017). Molecular data (based on cox1, nrLSU,
and nrSSU genes) have not support the generic status
of Janczewskia, as it is not monophyletic, and the two
investigated parasite species evolved independently
within the genus Laurencia with J. hawaiiana distantly
related to its host L. mcdermidiae, whereas J. morim-
otoi is nested within its host L. nipponica (Kurihara
et al., 2010). No other Janczewskia species have been
investigated genetically, and there are no genomic data
available.
For this study, we investigated the pigmented red
algal parasites Janczewskia tasmanica from Australia
and J. verruciformis from the Canary Islands, as prelim-
inary molecular data showed the presence of rbcL in
these parasites, indicating that photosynthetic capacity
may have been retained in contrast to the other three
known circular-­ mapping parasite plastid genomes
(Preuss et al., 2020; Salomaki et al., 2015; Salomaki &
Lane, 2019). The overall aims of this study were to: (i)
compare plastid genome architecture and characteris-
tics between parasites and their hosts and between all
parasites and (ii) investigate the evolutionary origin of
Janczewskia.
M ATER IAL S AN D M E T H ODS
Dried silica gel samples of two dark reddish pigmented
Janczewskia parasites and their associated hosts were
collected in Australia and the Canary Islands (Spain);
samples were also collected of one of the parasites
closest free-­living relatives (Table S1 in the Supporting
Information). DNA extraction of all samples followed a
modified cetyltrimethylammonium bromide protocol
(Zuccarello & Lokhorst, 2005) and was either used for
high-­throughput sequencing (HTS) or for Sanger se-
quencing to amplify individual genes.
For HTS, samples from the Canary Islands were
individually sequenced (n = 1), whereas samples from
Australia were pooled by combining each host (n = 1)
and parasite (n = 1–­ 5). Multiple parasites were com-
bined when possible due to their size. In addition, the
non-­host species Laurencia obtusa was sequenced,
as our preliminary results showed it to be a close rela-
tive of Janczewskia verruciformis. Libraries were con-
structed using Illumina TruSeq Nano DNA Library Prep
kits and were sequenced on a NovaSeq 6000 by Mac-
rogen Inc. (Seoul, Korea) with 150 bp paired-­end reads
and resulting in between 22,529,862 and 23,207,636
total read counts of raw data reads. Assembly and an-
notation of the plastid genomes followed as previously
described in Preuss et al. (2020). Briefly, sequenced
reads were processed in two separate workflows (i)
trimming and assembly with the CLC Genomic Work-
bench 20.0.05 (CLC Bio, Aarhus, Denmark) and (ii)
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952 |    PREUSS et al.

trimming in trimmomatic (Bolger et al., 2014) and then

508,866

1,263,035

799,726

2,949,102
501,479

1,601,509
assembly in SPAdes 3.14.0 (Prjibelski et al., 2020).
reads
Total Candidate contigs were identified using a custom data-
base containing known red algal genes, and annotation
was carried out in MFannot (Beck & Lang, 2010; https://
coverage

megas ​ u n .bch.umont ​ r eal.ca/cgi- ­b in/mfann ​ o t/mfann​

Mean

2585

1388
1091
otInt​erface.pl) and Aragorn (Laslett & Canback, 2004;

694
442

428
(X)

http://www.ansik​te.se/ARAGO​RN/) after circularity was

checked. Missing gene annotations identified through
(domain
Intron II

open reading frames (ORFs) and comparisons of anno-

tations between different species in MAUVE alignments
V)

1
1

1
were manually added in Geneious Prime 2021.2.2
Plastid genome characteristics of the Janczewskia parasites and their associated host species, plus one closely related non-­host species.

ncRNA

(https://www.genei​ous.com/). Genes were annotated

as pseudogenes if there were frameshift mutations due
1

1
1

1
to insertion/deletions or mutations leading to stop co-
dons. Frame shift mutations in identified pseudogenes
rRNA

in J. tasmanica were checked using custom designed

3

3
3

3
primers and a touchdown PCR with an annealing
tmRNA

temperature of 50°C for nine cycles followed by a de-

crease in annealing temperature to 45°C for 25 cycles
1

1
1

(Table S2 in the Supporting Information).

tRNA

For Sanger sequencing, the cox1 and rbcL genes

29

29

29

29
29

29

were sequenced for various host and parasite combi-

nations. These samples were used to confirm para-
2 (moeB, ycf46)

2 (moeB, ycf46)
Pseudogenes

site and host samples from the HTS samples and as

a comparison to available data from other sequenced
Abbreviations: ncRNA, noncoding RNA; rRNA, ribosomal RNA; tmRNA, transfer-­messenger RNA; tRNA, transfer RNA.

Janczewskia species. The GazF1 and GazR1 primers

were used to amplify partial cox1 (Saunders, 2005),
(Ψ)

0
0

and various primer combinations (F2-­R1452, F7-­RrbcS

Start, F57-­rbclrevNEW, and F765-­R1442) were used to
Unclassified

amplify rbcL (Díaz-­Tapia et al., 2018; Freshwater & Rue-

ness, 1994; Gavio & Fredericq, 2002; Kim et al., 2010;
ORFs

Saunders & Moore, 2013; Wang et al., 2000). The an-

20

20

26
21
19

17

nealing temperature for all PCRs was 45°C, following

Preuss and Zuccarello (2018).
Protein coding

For the plastid genome phylogeny, a total of 193

genes (w/o

concatenated plastid protein sequences were extracted

ORFs)a

and aligned with all available closely related plastid ge-

194a

194a

195

194
194

195

nomes in the Rhodomelaceae (Table S3 in the Support-

ing Information) using MAFFT (Katoh & Standley, 2013)
content (%)

in Geneious. Vertebrata spp. were used as outgroups

following Díaz-­Tapia et al. (2019).
70.2

70.2
70.8

70.8

70.9
70.8

For the cox1 and rbcL alignment, over 300 sequences

A-­T

within Laurencia or the Laurencieae were downloaded

from GenBank. All unique sequences were selected
Plastome
size (bp)

173,808

172,933

176,465

173,028

based on maximum likelihood (ML) phylogenies in IQ-­

171,833
173,810

TREE (Nguyen et al., 2015), and the longest unique se-

quence per taxon was kept. Chondria spp. were used
as an outgroup for the single gene alignments based
Janczewskia tasmanica

Laurencia catarinensis

on Díaz-­Tapia et al. (2017). Samples sequenced using

(on L. catarinensis)
Janczewskia tasmanica

both HTS and Sanger sequencing produced identical

Including pseudogenes.
Laurencia obtusa
verruciformis
(on L. sp. A25)

sequences for each sample, and only one sequence

Laurencia elata
(on L. elata)

Janczewskia

was included in the phylogenetic analyses.

Suitable models of amino acid evolution were se-
TA B L E 1

Parasites

Non-­host

lected based on the Bayesian information criterion

Hosts

(BIC) using ModelFinder in IQ-­TREE (Kalyaanamoorthy

et al., 2017). The model JTTCDMut+F+I+G4 was used
a

PLASTID EVOLUTION IN JANCZEWSKIA
for the multi-­gene phylogeny. The model TIM3+F+G4
was used for the first, HKY+F+I for the second, and
TN+F+I+G4 for the third codon position in cox1. The
model TIM2+F+I+G4 was used for first and second
codon position, and K3Pu+F+I+G4 was used for the
third codon position, in rbcL. Maximum likelihood to-
pologies were constructed, and branch support was
determined using 1000 replicates for non-­parametric
bootstrap (Felsenstein, 1985), Ultrafast bootstrap
(Hoang et al., 2018), and Shimodaira–­ Hasegawa-­
approximate likelihood-­ ratio test (SH-­aLRT; Guindon
et al., 2010) analyses.
Monophyly and multiple independent origins of the
parasitic species were tested using the approximately
unbiased test (AU test) in IQ-­ TREE. An alternative
topology was created with three groups: parasites,
free-­living red algae, and outgroup in Mesquite v3.70
(Maddison & Maddison, 2021).
Key parameter settings (e.g., random starting seed
number) of phylogenetic analyses to increase data re-
producibility can be found in Table S4 in the Supporting
Information.
Synteny of the plastid genomes was first checked
using a progressive Mauve alignment (Darling
et al., 2004), and the plastid genomes were visualized
linearly in OGDRAW (Greiner et al., 2019).
For comparative analysis, plastid genomes were
ordered by genome size/gene content as functional
and physical genome reduction should reflect different
transitional stages. Unannotated tRNAs were added to
the plastid genome of Choreocolax polysiphoniae and
missing genes were manually checked to see if they
were present in C. polysiphoniae and Harveyella mira-
bilis. We reannotated any missing protein coding genes
by identifying their potential position and reading frame
from closely related species. The new reading frame
was translated to amino acids and added to the anno-
tation if BLAST hits were found.
RESU LT S
Plastid genome characteristics and
comparisons
Six complete and one draft plastid genome of three
parasite and host combinations and one close free-­
living relative were assembled. Two of the complete
parasite plastid genomes are from Janczewskia tas-
manica, which we observed growing on two different
host species: Laurencia elata and an unidentified Lau-
rencia sp. (here called A25; Table 1). The mean cover-
age of the assembled plastid genomes varied between
428 and 2585× and generated between 501,479 and
2,949,102 reads. The plastid genomes of the parasites
J. tasmanica and J. verruciformis are large, 172,933
and 173,810 bp, and gene rich, with 194 and 195 protein
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    | 953
coding genes, including all photosynthetic genes (Ta-
bles 1 and 2; Table S3). The plastid genomes of the two
samples of J. tasmanica were identical with a slight dif-
ference in their complete size (173,808 vs. 173,810 bp).
This is the very first time a gene-­rich plastid genome of
a parasitic red alga has been documented.
Comparison between the six complete plastid ge-
nomes of Janczewskia spp. and Laurencia spp. show a
conserved architectural structure with many similarities
including genome size (171,833–­176,465 bp), number
of genes (194–­195), AT bias, and gene order (Table 1,
Figure S1 in the Supporting Information). The plastid
genomes are so similar that 90% of all shared protein
coding genes (194) are the same length (Table S5 in
the Supporting Information). Despite the great simi-
larities between the plastid genomes of parasites and
free-­living algae, the protein coding genes ycf46 (un-
characterized ATPase family AAA domain-­containing
protein) and moeB (Molybdopterin biosynthesis protein)
are pseudogenes in J. tasmanica but not in J. verruci-
formis. In J. tasmanica, there is a frameshift mutation in
the moeB gene caused by an indel and a stop codon
produced by a transversion, and a frameshift caused
by an indel are present in ycf46 (Table 1). In addition,
the protein coding gene asnB (asparagine synthetase
B) is present in J. verruciformis (and L. obtusa) but not
in J. tasmanica or any other compared Laurencia spp.
(Table S5).
Tentative patterns of plastid genome
evolution in parasitic red algae
Plastid genomes were compared in order of decreas-
ing size and gene content using the two Janczews-
kia parasite species and previous data: Choreocolax
polysiphoniae (Salomaki et al., 2015), Harveyella mi-
rabilis (Salomaki & Lane, 2019), and Pterocladiophila
hemisphaerica (Preuss et al., 2020). Two new anno-
tated hypothetical proteins were identified (ycf38 and
ycf19), and four non-­ annotated genes (ycf21, rpl32,
rpl33, and rpl35) were added to the plastid genome
of C. polysiphoniae. In contrast, three genes (dnaB,
fabH, and rpl35) were missing in H. mirabilis and not
in the smaller plastid genomes of C. polysiphoniae and
P. hemisphaerica. All compared genes in the reduced
plastids had missing photosystem I and II genes, loss of
all the cytochrome b/f complex genes except for petF,
and loss of ATP synthase genes and Calvin–­Benson–­
Bassham cycle genes. There were conservation and
reduction in number of rpo, rps, and rpl genes in the
parasites with the most reduced plastids (Choreoco-
lax and Pterocladiophila; Table 2). The plastid genome
of J. tasmanica indicates the first potentially reductive
changes, as it contains two pseudogenes (moeB and
ycf46) that were intact in J. verruciformis yet missing in
the other parasites (Table 2). Major changes occurred
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954 |    PREUSS et al.

TA B L E 2 Comparison of protein coding genes in parasitic red algae ordered from largest plastid genome (i.e., largest number of genes)
to smallest (i.e., with fewest number of genes; Table 3).

Janczewskia Janczewskia Harveyella Choreocolax Pterocladiophila

verruciformis tasmanica mirabilis polysiphoniae hemisphaerica
CDS
Photosystem I
psa genes (11)
Photosystem II
psb genes (19)
Cytochrome b/f complex
pet genes (10) 1 (petF) 1 (petF) 1 (petF)
ATP synthase
atp genes (8)
RuBisCO
rbcL gene (1)
RNA polymerase
rpo genes (5) 4 4
Ribosomal proteins—­SSU
rps genes (19) 18 16 16
Ribosomal proteins— ­LSU
rpl genes (28) 26a 24 21
Other conserved ORFs
ycf genes (26) Ψ ycf45 6 3 3
Other genes (67)c Ψ moeB 28a (Ψ gltBb) 27 (Ψ gltBb) 23
Note: Genes are grouped based on functional groups shown in OGDRAW, and total number of genes are seen in brackets. Dark gray cells indicate all genes
are present and light gray cells indicate gene loss with the total number of remaining genes given. Blank region indicates no gene for that functional group was
found in the plastid genome. Pseudogenes are indicated by Ψ.
a
dnaB, fabH and rpl35 are lost in H. mirabilis but not in C. polysiphoniae and P. hemisphaerica.
b
Following Salomaki and Lane (2019).
c
asnB was excluded from the comparison as it is present in J. verruciformis and absent in J. tasmanica and some free-­living red algae.

in H. mirabilis: the plastid genome dramatically reduced
in size due to high gene loss, but synteny was main-
tained in the remaining genes compared to free-­living
red algae. Both C. polysiphoniae and P. hemisphaerica
showed structural rearrangements, along with gene
loss, in their plastid genomes, which were not observed
in Janczewskia and Harveyella plastid genomes.
Phylogeny confirms multiple
parasite origins in Laurencia
The 46,454 amino acid alignment of 193 genes in-
cluded 21 taxa, including Janczewskia tasmanica and
J. verruciformis and their Laurencia host species. The
ML phylogeny showed four main lineages within Lau-
rencieae that corresponded to two recognized genera
(Laurenciella and Palisada), an unnamed lineage (Lau-
rencieae sp.), and a lineage including both Laurencia
and Janczewskia. All nodes within the phylogeny were
fully supported except for one (L. catarinensis and J. tas-
manica). Alternative topologies rejected (p = 0.000) the
monophyly of Janczewskia and confirmed, in addition
to the phylogenies, that these species had evolved at
least twice within Laurencia (Figure 1). The plastid ge-
nomes of the two samples of J. tasmanica were dis-
tantly related to their two different host species, L. elata
and Laurencia sp. A25. Janczewskia verruciformis was
closely related to the non-­host species L. obtusa and
distantly related to its host L. catarinensis (Figure 1).
The 663 bp alignment of the cox1 gene contained
61 taxa including four different Janczewskia parasites
and their associated host species. The ML phylogeny
did not support the monophyly of Janczewskia, as the
parasites evolved independently multiple times within
Laurencia (Figure 2). Most Janczewskia species were
distantly related to their hosts, except for J. morimo-
toi which was closely related to its host L. nipponica.
The Australian parasite J. tasmanica was distantly
related to its two host species, as shown earlier,
and formed an unsupported grouping with two non-­
hosts (L. karachiana MK796229 and Laurencia sp.
KX258826). The European parasite J. verruciformis
was nested in a group with specimens identified as
L. obtusa (our data and KX258828) from France and
L. viridis (KF492757) from the Canary Islands, Spain,

PLASTID EVOLUTION IN JANCZEWSKIA
supported by non-­parametric and ultrafast bootstrap,
and distantly related to its host L. catarinensis. The
Hawaiian parasite J. hawaiiana grouped with boot-
strap support to an unidentified non-­host Laurencia
sp. (GU223893) and not its host species, L. mcder-
midiae (Figure 2).
The 1467 bp alignment of the rbcL gene contained
84 taxa including three Janczewskia parasites and
their host species, when available. A third host species
for J. tasmanica, Laurencia elata, was included, too.
The ML phylogeny supported that the three included
Janczewskia species evolved independently within
Laurencia (Figure 3). Janczewskia tasmanica formed
a strongly supported grouping with the non-­host spe-
cies L. karachiana (MK796228) from Pakistan. The
parasite was distantly related to L. elata, Laurencia
sp. A25, and L. tasmanica, which were closely related
to each other. The European parasite J. verruciformis
was nested within a clade of L. obtusa sequences
with strong phylogenetic support and was distantly
related to its host L. catarinensis. The Japanese par-
asite J. morimotoi formed a strongly supported group
with two specimens in GenBank submitted as “Chon-
drophycus spp.” (DQ787585, MH388594) from South
Korea and Japan, respectively (Figure 3). No rbcL
sequences from its host L. nipponica were available,
and therefore it is uncertain if these topologies are
congruent between mitochondrial and plastid data
sets.
D I SCUS S I O N
Evolution and taxonomic placement of
Janczewskia
Our study demonstrated, for the first time, that parasitic
red algae can contain a gene-­rich plastid genome, as
seen in Janczewskia verruciformis, which is compa-
rable to the standard plastid genome in free-­living red
algae. Despite the gene richness of the plastid genome
of J. tasmanica, evidence of early stages of degradation
TA B L E 3 Comparison of plastid genome features between five parasitic red algal species ordered from largest plastid genome with
highest number of genes to smallest.
Janczewskia Janczewskia
verruciformis tasmanica
Size (bp) 172,933 173,808–­173,810
A-­T content (%) 70.2 70.8
CDS 195 194
Pseudogenes 0 2
tRNAs 29 29
Rearrangements no no
Note: Gene order of free-­living red algae was compared to parasites and indicated if any rearrangements were present. Features included total plastid genome
size, A-­T content, protein coding genes (CDS) without unclassified ORFs, pseudogenes, transfer RNAs (tRNAs), and plastid genome rearrangements. A-­T
content increases with loss of CDSs.
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    | 955
was observed (two pseudogenes), and despite their
similarity, these two Janczewskia species evolved in-
dependently. Comparing all circular-­mapping parasite
plastid genomes shows some tentative evolutionary
changes that are indicative of an order of change in
these genomes during the transition from free-­living to
parasitic. Also, the studied Janczewskia species do not
support its generic status, as the parasites evolved in-
dependently within Laurencia.
Janczewskia was established based on the mor-
phological characters of J. verruciformis (Solms-­
Laubach, 1877). Molecular data do not confirm the
monophyly of the genus and show that all four inves-
tigated species (J. hawaiiana, J. morimotoi, J. tasman-
ica, and J. verruciformis) evolved independently within
Laurencia (Kurihara et al., 2010; this study). Placing
parasitic red algae into the genus of its closest rela-
tive to reflect their evolutionary origin instead of para-
sitic genera has become a common practice (Freese
& Lane, 2021; Gurgel et al., 2018; Preuss et al., 2017;
Preuss & Zuccarello, 2018; Preuss & Zuccarello, 2019;
Silva, 1991). While not all Janczewskia species have
been sequenced, we believe, based on available data
and to preserve the monophyletic status of Laurencia
that now contains several polyphyletic Janczewskia
spp., that the generic name for these parasites should
be changed. Also, transferring the generitype J. verru-
ciformis, based on our data, would leave the yet un-
sequenced species as generic orphans. The slight
possibility that a minority of these unsequenced orphan
parasite species growing on other genera (Acantho-
phora, Chondria, and Osmundea; Preuss et al., 2017)
might be transferred from Laurencia in the future is
of course a possibility, but it is clear that the genus
Janczewskia cannot be maintained. During this writ-
ing, morphological work proposed the transfer of three
Janczewskia species (J. gardneri, J. moriformis, and
J. lappacea) into a new parasitic genus, Heterojancze-
wskia (Nam, 2022). We believe these changes ignored
previous studies showing that Janczewskia was not
monophyletic (Kurihara et al., 2010) and was nested
in Laurencia. The shared morphological characters of
Harveyella Choreocolax Pterocladiophila
mirabilis polysiphoniae hemisphaerica
90,654 90,243 68,701
76.5 79.5 74.2
84 75 70
1 1 0
24 25 26
no yes yes

956 |    PREUSS et al.
the parasites with their host (e.g., J. gardneri) indicates
the share evolutionary origin with their host and are
not a separate genus as is often the case for red algal
parasites.
We propose that all species in the genus Janczews-
kia and Heterojanczewskia be transferred to the genus
Laurencia:
Laurencia australis M.Preuss, Diaz-­Tapia, Verbrug-
gen & Zuccarello nom. nov.
Replaced name: Janczewskia tasmanica Falkenb.,
in Schmitz and Falkenberg (1897), Die natürlichen
Pflanzenfamilien nebst ihren Gattungen und wichtig-
eren Arten insbesondere den Nutzpflanzen unter Mit-
wirkung zahlreicher hervorragender Fachgelehrten,
Teil 1, Abteilung 2, p. 432, fig. 243 C.
Heterotypic synonym: Janczewskia australis
Falkenb. ex Reinbold (1899) nom. ined.
Etymology: (Latin = southern) refers to the country
it is found in.
Note: The existence of Laurencia tasmanica Hook.f.
& Harv., in Harvey (1849) precludes the transfer of
Janczewskia tasmanica Falkenb. into Laurencia, which
would result in a later homonym.
Laurencia hawaiiana (Apt) M.Preuss, Diaz-­Tapia,
Verbruggen & Zuccarello comb. nov.
Basionym: Janczewskia hawaiiana Apt, Phycologia
26: 328, 329, figs. 1–­7 (Apt, 1987).
Laurencia janczewskii M.Preuss, Diaz-­Tapia, Ver-
bruggen & Zuccarello nom. nov.
Replaced name: Janczewskia gardneri Setch.
& Guernsey, in Setchell Univ. Calif. Publ. Bot. 6: 12,
13, 14, 21, pl. 1, figs. 4–­6; pl. 3, figs. 15, 16; pl. 5, fig.
25 (1914).
Homotypic synonym: Heterojansczewskia gardneri
(Setch. & Guernsey) K.W.Nam. Korean J. Environ. Biol
40: 305 (2022).
Etymology: “janczewskii” of Janczewski, in refer-
ence to the previous designated genus name Jancze-
wskia Solms-­Laubach (1877).
Note: The existence of Laurencia gardneri Hollenb.,
in Smith & Hollenberg (1943) precludes the transfer of
Janczewskia gardneri Setch. & Guernsey into Lauren-
cia, which would result in a later homonym.
Laurencia lappacea (Setch.) M.Preuss, Diaz-­Tapia,
Verbruggen & Zuccarello comb. nov.
Basionym: Janczewskia lappacea Setch., in Setch-
ell, Univ. Calif. Publ. Bot. 6: 14–­16, 21, 22; pl. 6, figs.
28–­32 (Setchell, 1914).
Homotypic synonym: Heterojansczewskia lappa-
cea (Setch.) K.W.Nam. Korean J. Environ. Biol 40: 305
(2022).
Laurencia meridionalis (M.T.Martin & Pocock)
M.Preuss, Diaz-­Tapia, Verbruggen & Zuccarello comb.
nov.
Basionym: Janczewskia meridionalis M.T.Martin &
Pocock, J. Linn. Soc. London, Bot. 55: 62, figs. 3a, b
(Martin & Pocock, 1953).
15298817, 2023, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jpy.13373 by Faculty Of Medicine, Library Chulalongkorn University, Wiley Online Library on [18/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Laurencia morimotoi (Tokida) M.Preuss, Diaz-­
Tapia, Verbruggen & Zuccarello comb. nov.
Basionym: Janczewskia morimotoi Tokida, J. Jap.
Bot. 21: 127, figs. 1–­6 (Tokida, 1947).
Heterotypic synonym: Janczewskia tokidae
Saito (1971).
Laurencia parasitica M.Preuss, Diaz-­ Tapia, Ver-
bruggen & Zuccarello nom. nov.
Replaced name: Janczewskia moriformis Setch.,
Univ. Calif. Publ. Bot. 6: 11, 12, 21, pl. 1, figs. 1-­3; pl. 3,
figs. 20, 21; pl. 4, figs. 22, 23; pl. 5, fig. 24 (1914).
Homotypic synonym: Heterojansczewskia mori-
formis (Setch.) K.W.Nam. Korean J. Environ. Biol 40:
305 (2022).
Etymology: (Latin = parasitic) refers to the parasitic
life mode of this alga.
Note: The existence of Laurencia moriformis
­Kützing (1865) precludes the transfer of Janczewskia
moriformis Setch. into Laurencia, which would result in
a later homonym.
Laurencia ramiformis (C.F.Chang & B.M.Xia)
M.Preuss, Diaz-­Tapia, Verbruggen & Zuccarello comb.
nov.
Basionym: Janczewskia ramiformis C.F.Chang &
B.M.Xia, Stud. Mar. Sin. 14: 120, 126, fig. 1: 1–­8, fig. 2:
1–­6 (Chang & Xia, 1978).
Laurencia solmsii (Setch. & Guernsey) M.Preuss,
Diaz-­Tapia, Verbruggen & Zuccarello comb. nov.
Basionym: Janczewskia solmsii Setch. & Guernsey,
in Setchell, Univ. Calif. Publ. Bot. 6: 9, 10, 11, 20, pl.
2, figs. 7, 8, pl. 3, figs. 17–­19; pl. 5, figs. 26, 27 (1914).
Laurencia teysmannii (Weber Bosse) M.Preuss,
Diaz-­Tapia, Verbruggen & Zuccarello comb. nov.
Basionym: Janczewskia teysmannii Weber Bosse,
Siboga-­Expeditie Monographie p. 348, fig. 133 (Weber-­
van Bosse, 1923).
Laurencia verruciformis (Solms-­Laubach) M.Pre-
uss, Diaz-­Tapia, Verbruggen & Zuccarello comb. nov.
Basionym: Janczewskia verruciformis Solms-­
Laubach, Mém. Soc. Natl. Sci. Nat. Math. Cherbg. 21:
207, 210, pl. 3: figs. 1–­18 (1877).
Plastid genome degradation and evolution
The discovery of gene-­rich, and barely reduced, plas-
tid genomes in red algal parasites is important as it
indicates that fine-­scale patterns of plastid genome
degradation could be seen in parasites of different evo-
lutionary ages or dependencies on their host species.
The first evolutionary change that can be observed, in
Laurencia australis (as Janczewskia tasmanica), is a
pseudogenization of two genes (moeB and ycf46). This
pseudogenization was not observed in L. verruciformis
(as J. verruciformis). In flowering parasitic plants, pre-
dominately ndh photosynthetic genes initially became
pseudogenes and were subsequently lost (Wicke
 15298817, 2023, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jpy.13373 by Faculty Of Medicine, Library Chulalongkorn University, Wiley Online Library on [18/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
PLASTID EVOLUTION IN JANCZEWSKIA     | 957

F I G U R E 1 Maximum likelihood topology of 193 concatenated plastid gene amino acid sequences from the two parasites Janczewskia
tasmanica and J. verruciformis and closely related species within the Rhodomelaceae. Host and parasite combinations are indicated by
arrows. Outgroups of Vertebrata spp. were removed to facilitate presentation. Scale bar represents substitution per site. Branch support
values are given as non-­parametric bootstrap/UFbootstrap/SH-­aLRT. Asterisk (*) represents 100% support.

F I G U R E 2 Maximum likelihood topology of the cox1 sequences of four Janczewskia species and their associated hosts. Newly
sequenced taxa are highlighted in bold, and arrows connect each parasite to its host species. Chondria spp. outgroups were removed to
facilitate presentation. Scale bar represents substitution per site. Branch support values are given as bootstrap/UFbootstrap/SH-­aLRT.
Values <85% bootstrap, <95% UFbootstrap, and < 80% SH-­aLRT test are not shown. Asterisk (*) represents 100% support.

et al., 2013; Yu et al., 2017). This gene family is not pre- The first two discovered pseudogenes, moeB and
sent in other red algal plastids (Lee et al., 2016; Preuss ycf46 in Laurencia australis, have been completely lost
et al., 2020, 2021). in the parasites Choreocolax polysiphoniae, Harveyella
 15298817, 2023, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jpy.13373 by Faculty Of Medicine, Library Chulalongkorn University, Wiley Online Library on [18/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
958 |    PREUSS et al.

F I G U R E 3 Maximum likelihood topology of the rbcL gene of three Janczewskia parasites and some of their associated hosts and
related species. New sequenced taxa are highlighted in bold, and arrows connect each parasite to its host species. The outgroup of
Chondria spp. were removed to facilitate presentation. Branch support values are given as bootstrap/UFbootstrap/SH-­aLRT. Values <85%
bootstrap, <95% UFbootstrap, and <80% SH-­aLRT test are not shown. Asterisk (*) represents 100% support.

mirabilis, and Pterocladiophila hemisphaerica (Pre-
uss et al., 2020; Salomaki et al., 2015; Salomaki &
Lane, 2019). The gene moeB produces an enzyme that
catalyzes ATP in the molybdopterin biosynthetic path-
way (Leimkühler et al., 2001), which is not connected to
photosynthetic capability, and this function might not be
essential in parasites. In contrast, ycf46 is predicted to
play a role in the uptake and utilization of CO2 during pho-
tosynthesis (Jiang et al., 2015). The loss of ycf46 in the
plastid genome is not unusual in free-­living red algae and
has been observed in the freshwater orders Balbiania-
les, Batrachospermales, and Thoreales (Cho et al., 2018;
Evans et al., 2019; Paiano et al., 2018). This could indicate
that the loss of ycf46 is not linked to parasitism but an
easily lost function. More gene-­rich plastids and plastids
with different levels of degradation from various parasitic
algae are needed to determine if there are any regular
patterns in gene pseudogenization and loss.
Multiple models for the order of gene loss in para-
sitic plants have been proposed, which overall show
more similarities than differences (e.g., Barrett &
Davis, 2012; Naumann et al., 2016: Wicke et al., 2016).
For example, a five-­stage model of gene loss has been
proposed, where ndh genes are lost first, followed
by photosynthetic genes (psa, psb, and pet), then
PEP genes (rpo), then ATP synthase (atp), and lastly

PLASTID EVOLUTION IN JANCZEWSKIA
other functions (Barrett & Davis, 2012). The model
of Barrett and Davis was then modified by allowing
for these five stages to overlap (Graham et al., 2017).
Keeping in mind that gene loss in not linear (Graham
et al., 2017), one gene per million years is estimated
to be lost in parasitic plants (broomrapes; Cusimano
& Wicke, 2016). Our plastid gene loss comparison
of parasitic red algae, while still preliminary, shows
gene categories associated with photosynthesis are
missing (except for petF), while reduced genomes
have less but still contain gene categories associated
with non-­ photosynthetic functions (e.g., ribosomal
proteins: rps, rpl; Table 2). So loss of autotrophic abil-
ity may be the initial change that is seen in the tran-
sition from free-­living to parasitism. Similar orders of
gene loss have been observed in plastid genomes of
autotrophic lineages of apicomplexans (e.g., Plasmo-
dium), chrysophytes, and diatoms (Kim et al., 2020).
The minimal plastid size, before complete loss of the
plastid genome, has been postulated in the highly re-
duced plastid genome of the chrysophyte “Spumella”
(Dorrell et al., 2019). Loss of all photosynthetic genes
in independently evolved parasites are an indication
that these parasitic systems can be used to identify
patterns of plastid degradation that are applicable to
other parasites evolved from photosynthetic lineages.
Laurencia australis and L.
verruciformis are obligate hemiparasites
Obligate parasites cannot live independently from their
host and can be further divided into hemiparasites,
which have retained their photosynthetic capability,
and holoparasites, which have lost photosynthesis
completely. Our study showed the pigmented para-
sites Laurencia australis and L. verruciformis retained
all photosynthetic genes and that these parasites are
potentially capable of carrying out photosynthesis inde-
pendently. Early culture work showed that Janczewskia
gardneri incorporated NaH14CO3 when attached and
unattached to its host Laurencia spectabilis (now Os-
mundea spectabilis), which led to the assumptions that
parasite plastids were photosynthetic (Court, 1980).
Later our understanding of parasite plastid origin
changed, when it was shown that parasites transferred
material, including plastids, into host cells via sec-
ondary pit connections, and it was assumed that the
host plastids were retained in the parasite cells (Goff
& Coleman, 1995) and that parasite plastids were lost
or non-­functional. Despite the photosynthetic capabil-
ity of the parasite, these organisms rely on the host to
complete their development as shown in J. morimotoi
(Nonomura, 1979). The presence of all photosynthetic
genes in the plastid genomes and pigmentation in the
two parasites L. australis and L. verruciformis suggests
that these are obligate hemiparasites but depend on
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    | 959
their host to complete their life cycle. This hemipa-
rasitic life strategy may be a common characteristic in
the early stages of many red algal parasites that then
evolve increased host dependency and subsequent in-
creased plastid genome reduction.
This is the first observation of not only gene-­rich
plastid genomes of red algal parasites but also the early
stages of plastid genome degeneration. Future studies
of other parasitic red algae may show patterns that are
useful for understanding the changes that occur in the
transition between free-­living and parasitic life strate-
gies in these fascinating organisms.
AU T H O R C O N T R I B U T I O N S
Maren Preuss: Conceptualization (lead); data cura-
tion (lead); formal analysis (lead); funding acquisition
(lead); investigation (lead); methodology (lead); project
administration (lead); resources (equal); software (lead);
validation (lead); visualization (lead); writing –­original
draft (lead); writing –­review and editing (lead). Pilar
Díaz-­Tapia: Data curation (supporting); funding acqui-
sition (supporting); resources (equal); writing –­review
and editing (supporting). Heroen Verbruggen: Fund-
ing acquisition (supporting); resources (equal); software
(supporting); writing –­review and editing (supporting).
Giuseppe C. Zuccarello: Funding acquisition (support-
ing); project administration (supporting); writing –­origi-
nal draft (supporting); writing –­review and editing (lead).
AC K N O​W L E​D G E​M E N T S
We thank Michael Wynne and Craig Schneider for
advice on nomenclature. This work was funded by a
Marsden Fast Start (Grant no 19-­ VUW-­006) by the
Royal Society Te Apārangi, the Australian Biological
Resources Study (Activity 4-­G046WSD) and Xunta de
Galicia (grants ED481D/ 2017/011 and 03IN858A2019-
­1630129). Phylogenetic analyses were performed on
the Rāpoi High-­Performance Computing Facility of Vic-
toria University of Wellington, New Zealand.
ORCID
Maren Preuss https://orcid.
org/0000-0002-8147-5643
Pilar Díaz-­Tapia https://orcid.
org/0000-0003-4680-4867
Heroen Verbruggen https://orcid.
org/0000-0002-6305-4749
Giuseppe C. Zuccarello https://orcid.
org/0000-0003-0028-7227
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S U PP O R T I N G I N FO R M AT I O N
Additional supporting information can be found online
in the Supporting Information section at the end of this
article.
Figure S1. Annotated plastid genomes including
protein coding genes and rRNAs (except for ORFs)
from Janczewskia tasmanica and one of its hosts
(Laurencia elata) and J. verruciformis and its host
(Laurencia catarinensis) and closest relative (Laurencia
obtusa). Host and parasite combinations are indicated
by arrows. No gene arrangements, or gene loss,
are found between any of the compared plastid
genomes. Pseudogenes are indicated with asterisks in
J. tasmanica.
Table S1. Location and GenBank Accession numbers
for all sequenced samples. # host species unknown
and letter a-­e indicate the different host and parasite
combinations.
15298817, 2023, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jpy.13373 by Faculty Of Medicine, Library Chulalongkorn University, Wiley Online Library on [18/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Table S2. Sequence of custom designed primer
sequences to check areas with frameshift mutations in
annotated plastid genomes in J. tasmanica.
Table S3. Accession numbers of plastid genomes,
cox1, and rbcL sequences used for phylogenetic
analyses in alphabetical order by species. # indicates
rbcL sequence was extracted from the complete plastid
genome of the red alga.
Table S4. Key parameter settings used in phylogenetic
analyses.
Table S5. Summary of all genes and their length, in bp,
present in plastid genomes excluding unclassified open
reading frames of Janczewskia parasites and related
are grouped alphabetically by protein coding genes,
rRNA, and tRNA. Lengths between species are in bold.
How to cite this article: Preuss, M., Díaz-­Tapia,
P., Verbruggen, H., & Zuccarello, G. C. (2023).
Gene-­rich plastid genomes of two parasitic red
algal species, Laurencia australis and L.
verruciformis (Rhodomelaceae, Ceramiales), and
a taxonomic revision of Janczewskia. Journal of
Phycology, 59, 950–962. https://doi.org/10.1111/
jpy.13373